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First published online 3 May 2006
doi: 10.1242/dev.02355
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Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
* Author for correspondence (e-mail: eisen{at}uoneuro.uoregon.edu)
Accepted 9 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Primary motoneuron, Secondary motoneuron, LIM homeodomain, Interneuron, Spinal motoneuron, pMN domain, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Edlund and
Jessell, 1999; Eisen,
1999; Jurata et al.,
2000; Lee and Pfaff,
2001; Shirasaki and Pfaff,
2002; Sockanathan,
2003; Tanabe and Jessell,
1996). The subtype identity of a motoneuron is defined by its
axonal projection out of the central nervous system (CNS) and the specific
muscle it innervates (Eisen,
1999; Pfaff and Kintner,
1998; Tosney et al.,
1995). Although many proteins have been implicated in motoneuron
formation and subtype specification, the precise roles of some of these
proteins remain unresolved. In this paper, we focus on the roles of two
related proteins, Islet1 and Islet2, in formation and specification of subtype
identity of spinal motoneurons in zebrafish.
Zebrafish have two types of spinal motoneurons, primary motoneurons and
secondary motoneurons (Myers,
1985), both of which are derived from the spinal cord motoneuron
progenitor (pMN) domain (Kimmel et al.,
1994; Park et al.,
2004). Primary motoneurons (PMNs) are born early in development,
around the end of gastrulation. Each PMN is individually identifiable based on
its cell body position, axonal trajectory and the muscle region it innervates,
providing the opportunity to study vertebrate motoneuron formation at the
single cell level (Eisen et al.,
1986; Myers et al.,
1986; Westerfield et al.,
1986). Here, we focus on two of the PMNs, MiP, which has a
dorsally projecting axon, and CaP, which has a ventrally projecting axon. In
contrast to PMNs, secondary motoneurons (SMNs) arise later in development and
are more numerous than PMNs (Myers,
1985; Myers et al.,
1986). Although some SMNs project dorsally and others project
ventrally (Myers et al., 1986;
Westerfield et al., 1986), it
is currently unclear whether SMNs also develop individually identifiable
subtypes. Because PMNs have thus far only been described in anamniote
vertebrates such as fish and frogs (Eisen,
1994), it is thought that SMNs more closely resemble the
motoneurons described in amniote vertebrates
(Kimmel and Westerfield,
1990).
LIM homeodomain (LIM-HD) protein family members are expressed by all
vertebrate motoneurons studied to date and play a prominent role in several
aspects of motoneuron development, including initial specification and
adoption of a particular subtype identity
(Curtiss and Heilig, 1998;
Eisen, 1999;
Jurata et al., 2000;
Lee and Pfaff, 2001;
Pfaff and Kintner, 1998;
Shirasaki and Pfaff, 2002;
Sockanathan, 2003;
Tanabe and Jessell, 1996).
LIM-HD proteins have two N-terminal protein binding LIM domains and one
C-terminal DNA binding homeodomain (Bach,
2000; Curtiss and Heilig,
1998). In mouse and chick, the LIM-HD protein Islet1 appears to be
pan-motoneuronal around the time motoneurons exit the cell cycle
(Ericson et al., 1992;
Tsuchida et al., 1994).
Slightly later, motoneurons express a related LIM-HD protein, Islet2
(Thaler et al., 2004;
Tsuchida et al., 1994).
Studies in mouse demonstrated that Islet1 is required for motoneuron
formation; in the absence of Islet1, there is widespread cell death in the
ventral spinal cord (Pfaff et al.,
1996). By contrast, Islet2 is only required for formation of
visceral motoneurons, although it is expressed at least transiently in all
mouse spinal motoneurons (Thaler et al.,
2004).
As in other vertebrates, zebrafish motoneurons express Islet1 and Islet2
(Appel et al., 1995;
Inoue et al., 1994;
Korzh et al., 1993;
Tokumoto et al., 1995). SMNs
express both of these proteins, but it is still unclear whether they are
co-expressed or are in distinct SMN populations
(Appel et al., 1995;
Inoue et al., 1994). Unlike
SMNs, the expression patterns of islet1 and islet2 have been
studied in great detail in PMNs (Fig.
1A). islet1 is expressed in all PMNs around the time they
exit the cell cycle (Appel et al.,
1995; Inoue et al.,
1994; Korzh et al.,
1993; Tokumoto et al.,
1995). MiPs transiently downregulate and then reinitiate
islet1 expression prior to axogenesis; these cells do not express
islet2 (Appel et al.,
1995). By contrast, prior to axogenesis, CaPs initiate expression
of islet2 and then downregulate expression of islet1
(Appel et al., 1995;
Inoue et al., 1994;
Korzh et al., 1993;
Tokumoto et al., 1995). The
end result of this dynamic pattern of islet gene expression is that
by the time of axon extension, MiPs express exclusively islet1 and
CaPs express exclusively islet2. This expression pattern leads to the
hypothesis that differential expression of Islet proteins specifies PMN
subtype. Consistent with this idea, previous work suggested that Islet2 is
required for CaP formation (Segawa et al.,
2001). However, the role of Islet1 in formation of zebrafish PMNs
and MiP subtype specification has not been previously explored.
We used morpholino antisense oligonucleotides
(Nasevicius and Ekker, 2000)
and mRNA misexpression to investigate the roles of Islet1 and Islet2 in
formation of zebrafish motoneurons. Here, we provide evidence that in
zebrafish, as in mouse (Pfaff et al.,
1996), Islet1 is required for PMN and SMN formation. However,
instead of apparently dying like mouse motoneurons that lack Islet1
(Pfaff et al., 1996),
zebrafish PMNs appear to change fate and develop as interneurons in the
absence of Islet1. Surprisingly, despite the highly regulated expression
patterns of Islet1 and Islet2 (Fig.
1A), our results suggest that these proteins have redundant
functions. We provide evidence that Islet2 can substitute for Islet1 early on
(during motoneuron formation) and later on (during specification of MiP
subtype identity). Our results are consistent with a model in which upstream
factors that regulate the differential expression of Islet proteins or factors
that act in parallel to them establish the differences between PMN
subtypes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
RNA in situ hybridization
RNA in situ hybridization was performed as described by Appel et al
(Appel et al., 1995). RNA
probes include islet1 and islet2
(Appel et al., 1995).
Immunohistochemistry and TUNEL labeling
The following primary antibodies (Abs) were used: monoclonal anti-Islet
(Korzh et al., 1993), which
recognizes Islet1 and Islet2 proteins (1:200; 39.4D5 Developmental Studies
Hybridoma Bank); polyclonal anti-GABA (1:1000, Sigma); monoclonal zn1 (1:200)
(Trevarrow et al., 1990);
monoclonal znp1 (1:1000) (Melancon et al.,
1997; Trevarrow et al.,
1990); polyclonal anti-Lhx4 (1:500; S.A.H. and J.S.E.,
unpublished); and polyclonal anti-Lhx3 (1:500; S.A.H. and J.S.E.,
unpublished). The following secondary antibodies from Molecular Probes were
used: goat anti-mouse Alexa-488 (1:1000), goat anti-mouse IgG1
Alexa-488 (1:500), goat anti-mouse IgG2a Alexa-488 (1:500), goat
anti-mouse IgG2b Alexa-546 (1:500), goat anti-rabbit Alexa-546
(1:1000) and goat anti-rabbit Alexa-488 (1:1000). Goat anti-mouse Cy5 (1:200)
from Jackson Laboratories was also used. Embryos were fixed for 3.5-4.0 hours
in 4% paraformaldehyde (PFA) and 1xFix Buffer
(Westerfield, 1995) at
4°C; blocked in 1xPBS, 5% NGS, 4 mg/ml BSA, 0.5% Triton X-100 for 1
hour at room temperature; incubated in primary antibody diluted in block
overnight at 4°C; washed at room temperature for 1.5 hours in PBS + 0.1%
Tween-20; incubated in secondary antibody diluted in block for 4 hours at room
temperature; and then washed for 1.5 hours at room temperature in PBS + 0.1%
Tween-20. Embryos were stored in 4% paraformaldehyde until analyzed.
For triple labeling with Islet Ab, Lhx4 Ab and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling), embryos were first labeled with Islet and Lhx4 Abs. Embryos were then post-fixed for 20 minutes at room temperature in 4% PFA. After post-fixation, embryos were washed three times for 5 minutes with PBST (1xPBS + 0.1% Tween-20). Proteinase K (10 mg/ml) diluted 1:5000 in water was used to permeabilize embryos for 1.5 minutes at room temperature. Embryos were fixed for 15 minutes at room temperature in 4% PFA and washed out of fix with PBST. Embryos were incubated for 1 hour in the dark on ice followed by one hour in the dark at 37°C in TUNEL solution (Roche). After incubation, embryos were washed four times for 10 minutes in PBST; the last wash was overnight at 4°C.
Microscopy
Images of embryos were captured on a Zeiss Axioplan compound microscope
equipped with a Zeiss Axiocam, or on a Zeiss Pascal confocal microscope. Adobe
Photoshop was used to adjust brightness and contrast of images.
RNA and morpholino injections
islet1 RNA (Appel et al.,
1995; Inoue et al.,
1994; Tokumoto et al.,
1995) and islet2 RNA
(Appel et al., 1995;
Tokumoto et al., 1995) were
transcribed using the mMessage Machine (Ambion) according to instructions.
One-cell stage embryos were injected with several nanoliters of 56 ng/µl
islet1 RNA or 61 ng/µl islet2 RNA for overexpression
experiments. islet1 RNA was reduced to 28 ng/µl for rescue
experiments, but islet2 RNA remained at 61 ng/µl.
To create embryos with reduced Islet1, two splice-blocking morpholinos were
designed by Gene Tools (Corvallis, Oregon) to the splice donor sites after the
second and third exons of islet1. islet1E2 began at position 208 of
islet1 cDNA at the end of exon 2
(5'-TTAATCTGCGTTACCTGATGTAGTC-3') and ended in the second intron
of islet1 genomic DNA. islet1E3 began at position 472 of
islet1 cDNA at the end of exon 3
(5'-GAATGCAATGCCTACCTGCCATTTG-3') and ended in the third intron of
islet1 genomic DNA. The sequence for islet1E2 MO differs
from the corresponding sequence in islet2 genomic DNA at the end of
exon 2 (5'-GATTACGTACGGTACGAGCAACTAT-3') by 13 bp. The end of exon
3 in islet2 genomic DNA sequence
(5'-ATCCCAGGTAGTAGTAAAAATAATA-3') is different from
islet1E3 MO sequence by 18 bp. Several nanoliters of 1 mg/ml
islet1E2 and 1 mg/ml islet1E3 were co-injected into one-cell
stage embryos as described previously
(Lewis and Eisen, 2001).
Embryos looked generally healthy and had little or no Islet1 protein
remaining. The same phenotype was observed when an islet1 translation
blocking MO beginning at position –25 in the 5'UTR
(5'-CCCATGTCAAGAAAGTAAGGCGGTG-3') was injected into one-cell stage
embryos.
To create embryos with reduced Islet2, a translation blocking morpholino
was designed by Gene Tools to the translation start site. islet2 MO
began at position –2 of islet2 5'UTR
(5'-GGATGCGGTAGAATATCCACCATAC-3') and was tagged with fluorescein.
Several nanoliters of 5 mg/ml were injected into one-cell stage embryos as
describe previously (Lewis and Eisen,
2001). A second morpholino also designed to the translation start
site of islet2 gave the same phenotype as the first islet2
MO. The second islet2 MO began at position –9 of the
islet2 5'UTR (5'-GTAGAATATCCACCATACAGGAGGG-3').
Several nanoliters of 1 mg/ml islet1E2, 1 mg/ml islet1E3 and
5 mg/ml islet2 MOs were co-injected into one-cell stage embryos to
eliminate both Islet1 and Islet2 proteins.
Quantitation
We quantified the efficiency of our splice-blocking MOs by counting the
number of cell nuclei labeled with islet1 RNA adjacent to somites
8-11 and calculating the percentage of cells with nuclear islet1 RNA
labeling in islet1 MO-injected versus control (uninjected) embryos at
20-21 hpf. We also counted the number of cells in the pMN domain adjacent to
somites 8-11 labeled with Islet Ab at 28 hpf and calculated the percentage of
Islet-positive cells in islet1 MO-injected embryos versus control
embryos.
PMN axons stained with zn1 and znp1 Abs were counted in embryos at 24 or 28 hpf. Axons were counted as belonging to MiPs if they extended caudal and dorsal to the CaP cell body. Axons were counted as long CaP axons if they projected ventrally of the horizontal myoseptum. Axons were counted as short CaP axons if they exited the spinal cord, but did not project ventral of the horizontal myoseptum. To represent the number of axons in uninjected control versus injected embryos, we calculated the percentage of axons remaining in segments 8-12.
Islet Ab-labeled cells in the pMN domain adjacent to somites 8-10 were counted at 72 hpf from confocal microscopy images. We calculated the percentage of Islet-positive cells in MO-injected embryos in comparison with controls.
To count interneurons, we stained embryos with GABA Ab at 24 and 28 hpf and imaged them by confocal microscopy as described above. Cells were counted as in the KA'' position if they were in the medioventral spinal cord directly lateral to the floor plate. Cells were counted as in the V-K position if they were within three cell diameters dorsal of the floor plate. The average number of interneurons in the neural tube adjacent to segments 8-11 was compared between control and injected embryos.
To examine cell death, the number of cells co-expressing TUNEL and Lhx4 was counted in the ventral spinal cord adjacent to somites 8-11.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We also
stained embryos at 28 hpf with an Islet Ab that recognizes both Islet1 and
Islet2 proteins (Korzh et al.,
1993; Thor et al.,
1991). Islet Ab staining was absent from islet1
MO-injected embryos, indicating that Islet1 and Islet2 proteins were both
significantly reduced (Fig.
1D,E). The reduction in Islet2 protein could result from
islet1 MOs affecting the splicing of islet2 RNA. However,
this is extremely unlikely because the splice site sequences are very
different (see Materials and methods). In addition, islet2 mRNA was
absent from islet1 MO-injected embryos
(Fig. 2A,B). This contrasts
with the nuclear localization of islet1 mRNA in these embryos. These
results are consistent with the idea that our islet1 MOs are specific
for islet1, and raise the possibility that Islet1 regulates
islet2 expression or that PMNs are not specified in the absence of
Islet1 and thus do not express later PMN markers, such as islet2.
To examine the role of Islet1 in PMN formation, we looked for defects in
motor axon outgrowth at 24 and 28 hpf in islet1 MO-injected embryos
using zn1 and znp1 Abs, which recognize motoneurons
(Melancon et al., 1997;
Trevarrow et al., 1990). We
assayed the number of motor axons in segments 8-12 and found that both
dorsally projecting MiP and ventrally projecting CaP axons were significantly
reduced in islet1 MO-injected embryos
(Fig. 2D,E;
Table 1). islet1
MO-injected embryos had a few truncated CaP axons, something never seen in
control embryos (Table 1; data
not shown). However, the number of truncated CaP axons in islet1
MO-injected embryos was significantly fewer than the number of normal CaP
axons in control embryos. We examined the specificity of our islet1
MOs by co-injection of islet1 RNA and islet1 MO, and found
that PMN axons were restored and appeared normal
(Fig. 2F;
Table 1). Thus, we conclude
that Islet1 is required for zebrafish PMN formation. Interestingly, we also
found that, in most cases, injection of islet1 RNA did not restore
islet2 mRNA expression (Fig.
2C). These data suggest that that Islet1 does not regulate
islet2 mRNA expression and support the hypothesis that in the absence
of Islet1 PMNs are not specified, and thus do not express later markers, such
as islet2.
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). Essentially all SMN dorsally projecting and ventrally
projecting axons were absent from embryos injected with islet1 MO
(Fig. 3A,B), and the number of
Islet-positive cells in the ventral spinal cord was decreased by 52%
(Fig. 3A',B'). The
presence of Islet-positive cells in the ventral spinal cord of islet1
MO-injected embryos might result from reduction of MO efficacy as the embryos
developed over several days. Alternatively, it might be due to the presence of
Islet2 protein. We did two experiments to distinguish between these
possibilities. First, we injected one-cell stage embryos with a MO designed to
block the islet2 translation start site (islet2 MO) and
found that this caused a 10% reduction in the number of Islet-positive cells
in the ventral spinal cord (Fig.
3A,A',C,C'), an almost total loss of dorsally
projecting SMN axons and a partial loss and disorganization of ventrally
projecting SMN axons (Fig. 3C).
Second, we co-injected islet1 and islet2 MOs and found that
this caused a nearly complete loss of Islet-positive cells from the ventral
spinal cord (Fig. 3D,D').
islet1 MO-injected embryos had many more Islet-positive cells in the
ventral spinal cord than did embryos injected with both islet1 and
islet2 MOs; however, the severe reduction of SMN axon projections out
of the spinal cord was similar in both cases
(Fig. 3B,D). These data provide
evidence that the Islet Ab labeling of SMN cell bodies in islet1
MO-injected embryos was due to the presence of Islet2 protein. They also
support the idea that Islet1 protein is required for SMN formation and Islet2
protein is required for proper SMN axon outgrowth.
|
). By
contrast, our analysis of islet1 RNA expression in islet1
MO-injected embryos revealed that PMN cell bodies were still present in the
absence of Islet1 (Fig. 1B,C).
To confirm this, we examined cell death in the ventral spinal cord at 28 hpf
by co-labeling embryos with TUNEL and Lhx4 Ab, which labels PMNs as well as
some other neurons in the ventral spinal cord (S.A.H. and J.S.E.,
unpublished). The number of Lhx4-positive cells was the same in
islet1 MO-injected and control embryos, and we observed no increase
in the number of cells positive for TUNEL in experimental embryos, indicating
that PMNs did not die in the absence of Islet1
(Table 2).
|
;
Park et al., 2004), raising
the possibility that PMNs might develop as interneurons in the absence of
Islet1. We tested this hypothesis by examining co-expression of Lhx3 and zn1.
In control embryos, PMNs are the predominant cell type co-labeled by Lhx3 and
zn1 antibodies (Fig. 4A);
however, both of these antigens are also expressed by VeLD interneurons
(Appel et al., 1995) that are
derived from the pMN domain (Park et al.,
2004). islet1 MO-injected and control embryos had
approximately the same number and distribution of cells co-labeled with Lhx3
and zn1 Abs. In control embryos most Lhx3+, zn1+ cells
projected axons out of the spinal cord
(Fig. 4A), consistent with
their PMN identity. By contrast, in islet1 MO-injected embryos
Lhx3+, zn1+ cells did not project axons out of the
spinal cord, instead projecting axons within the spinal cord
(Fig. 4B), similar to
interneurons, and consistent with the hypothesis that in the absence of
Islet1, PMNs developed as interneurons.
|
;
Park et al., 2004). We counted
the number of cells in the VeLD, KA' and KA'' positions in the
spinal segments adjacent to somites 8-11. Cells were considered to be in the
KA'' position if they were located immediately lateral to the floor
plate. The number of cells in the KA'' position was similar in control
and islet1 MO-injected embryos
(Fig. 4C,D;
Table 3). Cells in the VeLD and
KA' positions were counted together because, although both cell types
are located dorsal to KA'' neurons
(Park et al., 2004) and within
three cell diameters dorsal of the floor plate, they are hard to distinguish
without additional markers. Thus, we refer to cells in this location as V-K.
There were significantly more cells in the V-K position of islet1
MO-injected embryos than in controls (Fig.
4C,D; Table 3).
Interestingly, although an average of 24 PMNs were lost from the spinal cord
adjacent to somites 8-11 of islet1 MO-injected embryos, there were
only an average of 11 extra GABA-positive cells in the V-K position
(Table 3), suggesting that some
PMNs did not develop into GABA-expressing interneurons. To determine whether
this was the case, we co-labeled embryos with GABA and zn1 Abs. At 28 hpf
these antigens co-localized in a few cells in the V-K position in control
embryos (Fig. 4E).
islet1 MO-injected embryos had more cells in the V-K position that
co-expressed GABA and zn1 (Fig.
4F). However, some of the zn1-positive interneuron-like cells did
not co-express GABA (Fig. 4F),
suggesting that some PMNs developed into a type of interneuron distinct from
VeLDs or KA' neurons. Alternatively, these cells may have adopted a
hybrid identity in which they developed interneuron-like axonal projections,
without expressing interneuron markers. Without interneuron type-specific
markers, we are unable to distinguish between these possibilities. Together
these data provide evidence that islet1 MO-injected embryos have more
pMN domain-derived interneurons than control embryos, and suggest that these
supernumerary cells are PMNs that have become interneurons in the absence of
Islet1.
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).
CaP subtype specification is independent of Islet2
The specific expression of Islet2 in CaPs, but not in MiPs, led us to
hypothesize that Islet2 is required for CaP subtype identity. CaPs transiently
co-express islet1 and islet2, but they downregulate
expression of islet1 prior to axogenesis
(Appel et al., 1995). Recent
characterization of narrow somite mutants revealed that PMNs that maintain
co-expression of Islet1 and Islet2 develop a CaP axon trajectory
(Lewis and Eisen, 2004),
suggesting that Islet2 is sufficient to cause a PMN to become a CaP, even when
Islet1 is not downregulated. Therefore, we asked whether misexpression of
Islet2 could turn MiPs into CaPs. We found that misexpression of
islet2 RNA had no effect on formation of dorsally projecting MiP
axons (Fig. 6A,B). Thus, MiPs
maintained their subtype identity despite co-expressing Islet1 and Islet2,
providing evidence that Islet2 is not sufficient to turn a MiP into a CaP.
|
);
therefore, we were able to use Islet Ab staining after 15 hpf to assay loss of
Islet2 in CaPs. Islet protein was absent from CaP cell bodies in
islet2 MO-injected embryos, indicating the MO was able to knock down
Islet2 protein (Fig. 6C,D).
islet2 MO-injected embryos had some CaPs with truncated axons and
some with abnormally branched axons; however, many CaPs were normal. These
results suggest that Islet2 is not required for formation of the ventral axon
that defines the CaP subtype identity, but that it is involved in later
aspects of CaP axon pathfinding. Together with our finding that
islet1 RNA can rescue all PMNs in islet1 MO-injected embryos
even when islet2 mRNA expression is not induced
(Fig. 2A-C), this result
suggests that Islet1 alone is sufficient for specification of CaP subtype
identity.
Islet2 can promote motoneuron formation and substitute for Islet1 in MiP formation
Our observation that Islet1 alone was sufficient for specification of CaP
subtype identity prompted us to ask whether Islet2 was similarly sufficient to
specify CaP. We tested whether Islet2 could promote CaP formation in the
absence of Islet1 by misexpressing islet2 RNA in islet1
MO-injected embryos and labeling them with zn1 and znp1 Abs. We found that CaP
formation was restored in these embryos
(Fig. 7A,B;
Table 1), revealing that,
similar to Islet1, Islet2 is sufficient to specify CaP subtype identity. These
results also reveal that Islet2 can substitute for Islet1 in motoneuron
formation.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Islet1 promotes motoneuron formation at the expense of interneuron formation
Zebrafish Islet1 is required for both SMN and PMN formation, and appears to
mediate a switch between motoneuron and interneuron fates in the pMN domain.
This apparently contrasts with the reported role of Islet1 in mouse, to
promote motoneuron survival (Pfaff et al.,
1996). However, several additional studies raise the possibility
that in mouse and chick, Islet1 may also inhibit interneuron formation. For
example, transplanting neural tubes from Islet1-deficient mice into chicks
prevents the death of nascent motoneurons. These surviving cells express
interneuron markers (Thaler et al.,
2004), although it is unclear whether they project motoneuron-like
axons out of the spinal cord or interneuron-like axons within the spinal cord.
Similarly, mouse embryos with a targeted deletion of the Mnx family member Hb9
initially express Islet1 in nascent motoneurons, allowing these cells to
develop as motoneurons and extend axons out of the spinal cord. However,
Islet1 expression is very quickly extinguished in these mice, and motoneurons
express interneuron markers (Arber et al.,
1999; Thaler et al.,
1999), suggesting that both Hb9 and Islet1 may participate in
inhibiting interneuron formation. Together, these results support the idea
that in mouse and chick, as in zebrafish, Islet1 may play a role in inhibiting
interneuron formation.
Whether Islet1 normally mediates a decision between motoneuron and
interneuron fates may depend whether these cells are derived from the same
progenitor population. Although lineage studies in chick using recombinant
retroviruses provided evidence that an individual spinal cord progenitor cell
can generate both motoneurons and interneurons
(Leber et al., 1990), more
recent studies in both chick and mouse have advocated the idea that
motoneurons arise from the pMN domain, whereas interneurons are generated from
adjacent p3 and p2 domains, as well as from other domains that are more distal
from the pMN domain (Briscoe and Ericson,
2001; Briscoe et al.,
2000). In mouse and chick, pMN domain-derived motoneurons
co-express Lhx3 and Islet1, whereas V2 interneurons, which are derived from
the p2 progenitor domain situated just dorsal to the pMN domain, express Lhx3
but not Islet1 (Ericson et al.,
1992; Sharma et al.,
1998; Tanabe et al.,
1998). Studies in the chick spinal cord show that misexpression of
Islet1 alone has no effect on motoneuron formation, whereas misexpression of
Lhx3 alone promotes V2 interneuron formation
(Tanabe et al., 1998;
Thaler et al., 2002).
Misexpression of both Lhx3 and Islet1 causes cells to become motoneurons, even
when they do not originate from the pMN domain
(Thaler et al., 2002). As in
mouse and chick, zebrafish PMNs co-express Islet1 and Lhx3, whereas VeLD
interneurons express Lhx3 but not Islet1
(Appel et al., 1995). However,
in contrast to mouse and chick, PMNs and VeLD interneurons are both derived
from the pMN domain (Park et al.,
2004). Clonal analysis in zebrafish reveals that a single ventral
neural tube progenitor in the pMN domain can generate PMNs, interneurons, or
both PMNs and interneurons; however, there is no consistent lineage
relationship among these cell types
(Kimmel et al., 1994;
Park et al., 2004). Zebrafish
lacking Islet1 lack PMNs, but have a normal number of Lhx3+ pMN
domain cells, consistent with the idea that loss of Islet1 results in pMN
domain-derived cells that express only Lhx3 and therefore develop as
interneurons. In contrast to chick, misexpression of zebrafish Islet1 alone
leads to formation of supernumerary PMNs. However, these cells only form in
the normal PMN position, suggesting that Lhx3+ cells within the pMN
domain become PMNs when they co-express Islet1. Thus, we suspect that in
zebrafish the fate decision between PMNs and interneurons is determined by the
interaction of transcription factors, such as Islet1, that are
motoneuron-specific within the pMN domain and Lhx3, which is expressed by both
motoneurons and interneurons. This is similar to what Thaler and colleagues
proposed happens in chick (Thaler et al.,
2002), except that in zebrafish the fate decision appears to occur
between cell types generated within the same progenitor domain, whereas in
chick it appears to occur between cell types generated in adjacent progenitor
domains. If our interpretation is correct, then Islet1 may only normally
mediate a switch between motoneuron and interneuron fates in cells that
co-express Lhx3 and are derived from the same progenitor population.
An outstanding question that remains to be addressed is the identity of the
supernumerary interneurons that form in zebrafish in the absence of Islet1.
The pMN domain generates at least four types of interneurons: VeLD, KA',
KA'' and CiD (Park et al.,
2004). Unfortunately, we currently have few markers other than
cell morphology to distinguish these cells
(Lewis and Eisen, 2003). Using
GABA as a marker for VeLD, KA' and KA'' interneurons, we found that
the number of cells in the V-K position increased in islet1
MO-injected embryos, whereas the number of cells in the KA'' position was
unchanged. Interestingly, the number of GABA-positive, supernumerary
interneurons in islet1 MO-injected embryos was about half the number
of PMNs that were lost, raising the possibility that some PMNs were only
partially transformed into interneurons, and changed their axon trajectory
without expressing GABA. Alternatively, there might be an increase in another
type of pMN domain-derived interneuron that was not detectable with our
markers, or there could have been an increase in several types of
interneurons, only some of which express GABA. We are unable to distinguish
among these possibilities with the available interneuron markers. Thus, it is
crucial to identify cell-type specific markers for pMN domain derivatives to
further assess the fates of these cells under different conditions.
Islet1 and Islet2 are functionally redundant
Previous studies have suggested that Islet1 and Islet2 may have redundant
functions during motoneuron formation; however, this has not previously been
tested. Thaler and colleagues (Thaler et
al., 2004) proposed that the level of Islet protein, not the
specific type of Islet protein, determines whether a cell becomes a visceral
motoneuron. We have tested directly whether Islet1 and Islet2 have redundant
functions by co-injecting embryos with islet1 MO and islet2
RNA to learn whether Islet2 can substitute for Islet1 during motoneuron
formation in zebrafish. We found that Islet2, like Islet1, could promote
motoneuron formation, consistent with the hypothesis that the differences
between Islet1 and Islet2 proteins are unimportant for motoneuron
formation.
CaP and MiP subtype specification is independent of the differences between the Islet1 and Islet2 proteins
What is most surprising is that our results provide evidence that, despite
the exquisite and dynamic regulation of expression of islet1 and
islet2 in zebrafish PMNs (Appel et
al., 1995; Inoue et al.,
1994; Korzh et al.,
1993; Tokumoto et al.,
1995), the differences between these proteins are not important in
establishing the differences between the different PMN subtypes. Islet2 is
expressed only in CaPs, yet our data suggest that either Islet1 or Islet2 is
sufficient for specification of CaP subtype identity. Previous studies from
our laboratory suggested that Islet2 expression might force PMNs to develop as
CaPs, because in some mutants, PMNs expressing both Islet1 and Islet2 formed
CaP axon projections and not MiP axon projections
(Lewis and Eisen, 2001). Thus,
it was surprising that misexpression of islet2 RNA did not prevent
formation of MiP dorsal projections, indicating that in the context of
wild-type embryos, Islet2 is insufficient to inhibit MiP development. We also
found that knockdown of Islet2 protein resulted in only minor defects in CaP
axon outgrowth. These results contrast with a previous study showing that
expression of a dominant negative Islet2 LIM domain caused severe defects in
CaP projections and in some cases caused CaPs to develop into interneurons
(Segawa et al., 2001).
However, the same study found that Islet2 knockdown using MOs resulted in a
much less severe effect on CaPs that appears to be very similar to what we
have described. One possible way to reconcile these results is to imagine that
the dominant-negative Islet2 LIM domain interfered with some, but not all
Islet1 functions, consistent with the finding of Thaler and colleagues
(Thaler et al., 2002) that LIM
domains of different LIM-HD proteins can have overlapping and non-overlapping
functions. If this were the case, it could significantly lower the efficacy of
both Islet2 and Islet1 proteins, resulting in insufficient Islet function to
repress interneuron formation. This would then be similar to the result we got
from knocking down Islet1 alone, and fits well with the model that the overall
levels of Islet protein are important in motoneuron formation
(Thaler et al., 2004).
Together, these results lead to the surprising conclusion that Islet2 is not
required for CaP subtype identity, despite its specific expression in CaP
motoneurons.
Islet1 expression is maintained in MiPs but not in CaPs; therefore, we hypothesized that this late expression of Islet1 is required for MiP subtype identity. However, when we substituted Islet2 for Islet1 in embryos co-injected with islet2 RNA and islet1 MOs, MiPs formed normal, dorsally projecting axons. These results do not support our original hypothesis, but instead indicate that Islet1 protein is not required for MiP subtype specification if another Islet protein is available.
There have been previous reports that highly related proteins can
substitute for one another, despite their distinct expression patterns
(Geng et al., 1999;
Hanks et al., 1995;
Hirth et al., 2001;
Wang and Jaenisch, 1997;
Wang et al., 1996). Sequence
analysis of zebrafish Islet1 and Islet2 proteins indicate they are highly
related [98% identity in the DNA-binding homeodomain and 92% or 70% identity
in the first and second LIM domains, respectively; Tokumoto et al.
(Tokumoto et al., 1995)]. Our
data show that Islet1 and Islet2 are also able to substitute for one another
functionally during motoneuron formation, suggesting that the regulation of
islet1 and islet2 transcript expression, rather than the
distinct sequences of the proteins they encode, establishes their specific
functions. Therefore, transcription factors expressed very early in motoneuron
development are likely determinants of PMN subtype identity. islet1
is the earliest reported gene expressed in PMNs following expression of
so-called patterning genes, such as olig2
(Park et al., 2002) and
nkx6.1 (Cheesman et al.,
2004), that are expressed in pMN domain progenitor cells as well
as in post-mitotic PMNs (Cheesman et al.,
2004; Park et al.,
2002). Thus, it will be important to determine whether any of the
known patterning genes, or patterning genes yet to be discovered, plays a role
in PMN subtype specification by regulating islet expression.
Islet proteins function in motoneuron development in other taxa
Islet1 appears to be expressed in all vertebrate motoneurons and in every
instance in which it has been examined, it seems to be necessary for their
formation. By contrast, the single islet gene of the fruit fly,
Drosophila melanogaster, is expressed only in a subset of motoneurons
that project their axons ventrally (Thor
and Thomas, 1997) and thus cannot be required to confer
`motoneuron-ness' (Thor and Thomas,
2002). Similar to zebrafish, in the absence of Islet function
Islet-expressing fruit fly motoneurons are present, but their axonal
projections are aberrant. In most cases, the cells still send axons into the
periphery, but they fail to make appropriate neuromuscular connections.
Consistent with this, overexpression of Islet also causes some motoneurons to
project to inappropriate muscles. Interestingly, however, two Islet-positive
fruit fly motoneurons apparently do not project axons into the periphery in
the absence of Islet function, but instead project axons within the CNS, in
essence acting as though they have become interneurons, similar to what we
have reported for zebrafish PMNs in the absence of Islet1. No islet
homolog has been reported in the nematode worm, Caenorhabditis
elegans. However, three related LIM-HD genes, lin-11
(Hobert and Ruvkun, 1998),
lim-6 (Hobert et al.,
1999) and lim-4
(Tsalik et al., 2003),
function in aspects of development of specific C. elegans
motoneurons: axon pathfinding in the case of lin-11, neurotransmitter
receptor expression in the case of lim-4 and axon pathfinding and
neurotransmitter synthesis in the case of lim-6. Thus far, mouse is
the only species in which a LIM-HD protein, in this case Islet1, appears
required for motoneuron survival (Pfaff et
al., 1996). Other LIM-HD proteins are expressed in tetrapod
vertebrate motoneurons (Sharma et al.,
1998; Tsuchida et al.,
1994), but like the LIM-HD proteins of flies, worms and zebrafish,
these all seem to function in later aspects of motoneuron development,
especially axon pathfinding and neurotransmitter choice. Thus, it will be
important to study motoneuron development and LIM-HD protein function in other
species to fully understand how Islet1 function has changed over time.
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ACKNOWLEDGMENTS |
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REFERENCES |
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